ABSTRACT These studies aim to define at a higher level of resolution the mechanism by whch the Retinal Determination Gene Network or pathway (RDGN) governs breast cancer onset and progression. Hyperactive growth factor receptor signaling remains active in many tumors that resist current therapies. The Drosophila dac gene was cloned as a dominant inhibitor of the hyperactive EGFR (Elipse). DACH1 reversed the transformed phenotype of mammary epithelial cells in 3-dimensional culture, inhibited oncogene-mediated breast tumorigenesis, blocked breast cancer epithelial cell DNA synthesis, colony formation, growth in matrigel, inhibited epithelial mesenchymal transition (EMT), tumor growth and metastasis in mice. Genetic deletion of Dach1 in the mouse results in perinatal lethality therefore we developed conditional Dach1 knockout tri- transgenic systems. Our studies provide support for a model in which DACH1 physical interactions with specific proteins coordinate DACH1-tumor suppression. These interactions govern growth suppression in breast tumor genetic subtype specific manner. DACH1 binds p53 to enhance p53 tumor suppressor functions. DACH1 binds and inhibits the function of growth inducing proteins through distinct mechanisms (YB-1, EYA1, FKHR) (Fig. 1). These studies will further characterize a novel tumor and metastasis suppressor pathway. We hypothesize that inactivation of the DACH1/EYA pathway is a key signaling event contributing to mammary tumorigenesis and metastasis. We will determine the mechanism by which DACH1 inhibits breast tumor cellular proliferation and metastasis in vivo. Photo-uncaging to induce single cell level Cre excision will allow determination of sister cell interactions and the in vivo significance of a new model of tumor suppression. Functional analyses of DACH1-secreted factors and synthetic lethal screens will identify new cancer targets.